It has been demonstrated in this lab that infectious, non-propagating replicon particles from Venezuelan equine encephalitis virus (VEE) act as an adjuvant when co-delivered parenterally with antigen. These VEE replicon particles (VRPs) not only amplify the existing systemic antibody response to an antigen, but also induce a mucosal IgA response and a CD8 T cell response, both of which are undetectable in the absence of VRP. Because VRPs are so effective at inducing robust immunity in multiple arms of the immune system, we are pursuing these particles as an adjuvant that could be included in a wide range of vaccines. Such an adjuvant has the potential to induce protection against diseases for which vaccines have so far proven ineffective. In order to better understand the mechanism by which VRPs act as an adjuvant, it is the goal of the work proposed here to investigate the role of dendritic cells (DCs) in the VRP-mediated adjuvant effect. It has been demonstrated that VRPs infect DCs both in vitro and in vivo, and in doing so trigger upregulation of dendritic cell costimulatory molecules and secretion of inflammatory cytokines. Because DCs are central to the link between the innate and adaptive immune response, we suspect that VRP interactions with DCs are key to their adjuvant effect. We will identify the DC subsets recruited to the draining lymph node of mice after immunization with VRPs, and will also determine which subsets are infected by VRPs. We have produced an in vitro system in which antigen-pulsed bone-marrow derived DCs can be infected with VRPs, which results in enhanced IL-2 production by antigen-specific CD8 T cells. In this proposed research we will identify cytokines produced by dendritic cells in response to VRP-infection, using both bone-marrow derived DCs and DC subsets isolated from mice. We will assess the effect of these VRP-infected DCs on activation and cytokine production of OVA-specific CD4 and CD8 T cells derived from OT-II and OT-1 TCR-transgenic mice. We have shown that secreted factors from VRP-activated DCs amplify the T cell response to antigen, and we will determine which DC-secreted factors are involved in this effect. This will be accomplished by blocking cytokines with antibodies, and also by using DCs from cytokine-knockout mice, or T cells from cytokine receptor knockout mice. We will confirm the in vivo relevance of these cytokines to the adjuvant effect, either by in vivo cytokine blocking or by use of knockout mice. PUBLIC HEALTH RELAVENCE: The goal of this research is to identify mechanisms by which the immune response to a vaccine target can be enhanced. Such findings can potentially be applied to any vaccine target, enabling the development of more effective vaccines.